Peptides capable of modulating inflammatory heart disease

ABSTRACT

Disclosed are novel peptides that modulate inflammatory heart disease. Also disclosed are DNA molecules encoding the peptides, and methods of making the peptides.

BACKGROUND

1. Field of the Invention

This invention relates to novel peptides, and to genes encoding thepeptides, which are capable of inducing inflammatory cardiomyopathy invivo.

2. Related Art

Cardiovascular diseases are a major cause of death in Western societies.Various risk factors have been associated with the pathogenesis ofcardiovascular disease, including such factors as high cholesterollevels, smoking, stress, high blood pressure, obesity, andhyperglycemia. Recent evidence suggests that certain bacterialinfections may be a causative event in the development of certain heartdiseases (Danesh et al., Lancet, 350: 430-436 1997!). In particular,Chlamydia infections have recently been shown to be linked, bothepidemiologcally and experimentally, to heart disease (Danesh et al.,supra; Ossewaarde et al., Epidemiol. and Infect., 120: 93-99 1998!).

Inflammatory heart disease and dilated cardiomyopathy similar to thatwhich occurs in humans can be induced in mice by immunization of themice with myosin protein obtained from heart muscle (Neu et al., J.Immunol., 139: 3630-3636 1987!). Immunization of Balb/c mice with apeptide consisting of amino acids 614-643 of cardiac alpha-myosin caninduce inflammatory heart disease in the mice (Pummerer et al., J. ClinInvest., 97: 2057-2062 1996!).

In view of the prevalence and devastating effects of cardiovasculardisease, it would be beneficial to identify compounds that decrease therisk of inflammatory cardiomyopathy and methods of decreasing orpreventing such disease.

Accordingly, it is an object of the present invention to provide novelcompounds that are useful in decreasing and/or preventing inflammatorycardiomyopathy. Other such objects will be readily apparent to theskilled artisan.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated nucleicacid molecule encoding a peptide capable of inducing inflammatorycardiomyopathy selected from the group consisting of the nucleic acidmolecules of SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

In another embodiment, the present invention provides a peptide capableof inducing inflammatory cardiomyopathy selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, and 16. Optionally, the peptide maybe acylated at the amino terminus, and preferably acylation is by meansof acetylation.

In yet another embodiment, the invention provides a vaccine comprisingthe peptide of SEQ ID NO:3 or SEQ ID NO:15. Optionally, the vaccine mayfurther comprise an oligodeoxynucleotide selected from the groupconsisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ IDNO:13.

In a further embodiment, the invention comprises a method of decreasingor preventing inflammatory cardiomyopathy comprising administering to amammal in need thereof the peptide of SEQ ID NO:3 or SEQ ID NO:15.

The invention further comprises a method of inducing inflammatorymyocarditis in a mammal comprising administering to the mammal thepeptide of any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:16.

Yet further, the invention comprises a method for measuring the risk ofinflammatory cardiomyopathy in a mammal, comprising incubating a sampleof the mammal's T-cells with the peptide of any of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,and SEQ ID NO:16.; measuring the proliferation of the T-cells; andcomparing proliferation of the T-cell sample with control cells, whereina higher level of proliferation as compared to control cells indicatesan increased risk of inflammatory cardiomyopathy.

Still further, the invention comprises an isolated nucleic acid moleculecomprising the nucleic acid molecule of SEQ ID NO:25 or SEQ ID NO:26,and peptides encoded by such nucleic acid molecules such as those of SEQID NO: 25 or SEQ ID NO:26.

The invention further provides vaccines comprising the peptides of SEQID Nos: 2, 4, 5, 6, 7, 8, 9, and 16.

Still further, the invention provides a method for measuring the risk ofinflammatory cardiomyopathy in a mammal, comprising measuring the levelof antibodies directed against one or more inflammatory cardiomyopathypeptides in the serum of the mammal, and comparing such antibody levelto that contained in control serum, where an increase in antibodies ascompared with control serum indicates an increased risk of inflammatorycardiomyopathy.

DETAILED DESCRIPTION OF THE INVENTION

Included in the scope of the present invention are novel peptides thatcan induce inflammatory heart disease in vivo. DNA molecules encodingthese peptides, methods for preparing these peptides, and methods ofusing these peptides. Also within the scope of the present invention arenovel peptides which decrease, or prevent inflammatory cardiomyopathy,as well as DNA molecules encoding these peptides, methods for preparingthese peptides, and methods of using these peptides.

Also contemplated within the scope of the present invention arenon-human mammals in which inflammatory cardiomyopathy has been inducedvia administration of one or more of the peptides of the presentinvention. Such non-human mammals are preferably chimpanzees or monkeys,pigs, dogs, rabbits, goats, sheep, or rodents such as mice or rats. Mostpreferred are rabbits, mice, and rats.

The scope of the present invention further includes an assay fordetermining the risk of inflammatory cardiomyopathy wherein blood isscreened for the presence of activated T-cells and/or circulatingantibodies directed to the novel peptides of the present invention.

The scope of the present invention further includes certainoligodeoxynucleotides that act as potent stimulators of the immunesystem.

A fragment of the alpha-myosin chain protein has been identified thatcan induce autoimmune inflammatory cardiomyopathy in vivo. In contrast,a homologous region of beta-myosin chain protein does not induceautoimmune inflammatory cardiomyopathy, and has been shown to preventautoimmune inflammatory cardiomyopathy. An amino acid sequencecomparison of the homologous peptides of the alpha and beta chains ofmyosin indicates that the motif MAxxxS (SEQ ID NO:1) is important forpathogenicity in vivo. Surprisingly, a search of public databases usingSEQ ID NO:1 has identified various peptides from cysteine rich outermembrane proteins ("CRPs") of Chlamydia trachomatis that match the motifof SEQ ID NO:1. In addition, CRPs from Chlamydia psittaci and Chlamydiapneumoniae also share sequence homology to SEQ ID NO:1. As described inthe Examples herein, some of these peptides also induce inflammatorycardiomyopathy in vivo.

The term "inflammatory cardiomyopathy peptide" refers to any peptide ofthe present invention, including without limitation, the peptides of SEQID Nos: 2, 4, 5, 6, 7, 8, 9, and 16, that can induce inflammatorycardiomyopathy in mammals such as mice in vivo.

For purposes herein, "inflammatory cardiomyopathy" refers toinflammation of the heart as measured by an increased heart/body weightratio as compared with such ratio for normal hearts, and/or infiltrationof the heart tissue with mononuclear cells which are normally absent innormal heart tissue.

The term "therapeutic cardiomyopathy peptide" refers to any peptide ofthe present invention, including, without limitation, the peptides ofSEQ ID Nos: 3 and 15, that can reduce or prevent inflammatorycardiomyopathy in mammals such as mice in vivo, whether administeredalone or in combination with an inflammatory cardiomyopathy peptide.

The term "inflammatory cardiomyopathy peptide fragment" refers to apeptide that is less than 16 amino acids in length but which hasbiological activity consistent with that described herein forinflammatory cardiomyopathy peptides.

The term "therapeutic cardiomyopathy peptide fragment" refers to apeptide that is less than 14 amino acids in length but which hasbiological activity consistent with that described herein fortherapeutic cardiomyopathy peptides.

The term "inflammatory cardiomyopathy peptide variant" refers to aninflammatory cardiomyopathy peptide whose amino acid sequence containsone or more amino acid sequence substitutions, deletions, and/orinsertions as compared to the peptides of SEQ ID NOS 2 and 16. Preferredinflammatory cardiomyopathy peptide variants are those containingconservative changes or alanine at one or more of amino acid residues1-5, 8-10, and/or 12-16 as compared with SEQ ID NOS:2 and 16.

The term "therapeutic cardiomyopathy peptide variant" refers to antherapeutic cardiomyopathy peptide whose amino acid sequence containsone or more amino acid sequence substitutions, deletions, and/orinsertions as compared to the peptides of SEQ ID NOS 3 and 15. Preferredtherapeutic cardiomyopathy peptide variants are those which contain aconservative change or alanine at one or more of amino acid residues1-4, 8-9, and 12-13.

The term "inflammatory cardiomyopathy peptide derivative" refers to aninflammatory cardiomyopathy peptide, fragment, or variant that has beenchemically modified, as for example, by addition of one or morepolyethylene glycol, dextran, sugar, phosphate, Fc peptide, and/or othersuch molecules, where such molecule or molecules are not naturallyattached to the inflammatory cardiomyopathy peptides.

The term "therapeutic cardiomyopathy peptide derivative" refers to atherapeutic cardiomyopathy peptide, fragment, or variant that has beenchemically modified, as for example, by addition of one or morepolyethylene glycol, dextran, sugar, phosphate, Fc peptide, and/or othersuch molecules, where such molecule or molecules are not naturallyattached to the therapeutic cardiomyopathy peptides.

As used herein, the terms "nucleic acid molecule encoding aninflammatory cardiomyopathy peptide" and "inflammatory cardiomyopathynucleic acid" refer to a nucleic acid molecule or fragment thereof that(a) has the nucleotide sequence as set forth in any of SEQ ID NOS:2, 4,5, 6, 7, 8, 9, and 16; (b) has a nucleic acid sequence encoding abiologically active inflammatory cardiomyopathy peptide that is at least70 percent identical, but may be greater than 70 percent, i.e., 75, 80,85, 90, 95 percent, or even greater than 95 percent identical, to any ofthe peptides of SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, and 16; (c) is anaturally occurring allelic variant of (a) or (b); (d) is a nucleic acidvariant of (a)-(c) produced as provided for herein;(e) has a sequencethat is complementary to (a)-(d); and/or (f) hybridizes to any of(a)-(e) under conditions of high stringency.

As used herein, the terms "nucleic acid molecule encoding a therapeuticcardiomyopathy peptide" and "therapeutic cardiomyopathy nucleic acid"refer to a nucleic acid molecule or fragment thereof that (a) has thenucleotide sequence as set forth in any of SEQ ID NOS: 25 or 26; (b) hasa nucleic acid sequence encoding a biologically active therapeuticcardiomyopathy peptide that is at least 70 percent identical, but may begreater than 70 percent, i.e., 75, 80, 85, 90, 95 percent, or evengreater than 95 percent identical, to any of the peptides of SEQ IDNOS:3 or 15; (c) is a naturally occurring allelic variant of (a) or (b);(d) is a nucleic acid variant of (a)-(c) produced as provided forherein; (e) has a sequence that is complementary to (a)-(d); and/or (f)hybridizes to any of (a)-(e) under conditions of high stringency.

Percent sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two peptides or polypeptides, or one peptide and one polypeptide. Byway of example, using a computer algorithm such as BLAST, BLAST2, orFASTA, the two (poly)peptides for which the percent sequence identity isto be determined are aligned for optimal matching of their respectiveamino acids (the "matched span", which can include the full length ofone or both sequences, or a pre-determined portion of one or bothsequences). Each computer algorithm provides a "default" opening penaltyand a "default" gap penalty, and a scoring matrix such as PAM 250 (forFASTA) or BLOSUM 62 (for BLAST algorithms). A preferred algorithm isBLAST2.

A standard scoring matrix (see Dayhoff et al., in: Atlas of ProteinSequence and Structure, vol. 5, supp.3 1978!) can be used in conjunctionwith the computer algorithm. The percent identity can then be calculatedby determining the percent identity using an algorithm contained in aprogram such as FASTA: ##EQU1## Peptides or polypeptides that are atleast 70 percent identical will typically have one or more amino acidsubstitutions, deletions, and/or insertions as compared with wild typepeptide. Usually, the substitutions of the native residue will be eitherto alanine residues, or to conservative amino acids so as to have littleor no effect on the overall net charge, polarity, or hydrophobicity ofthe peptide. Conservative substitutions are set forth in Table I below.

                  TABLE I    ______________________________________    Conservative Amino Acid Substitutions    ______________________________________    Basic:               arginine                         lysine                         histidine    Acidic:              glutamic acid                         aspartic acid    Uncharged Polar:     glutamine                         asparagine                         serine                         threonine                         tyrosine    Non-Polar:           phenylalanine                         tryptophan                         cysteine                         glycine                         alanine                         valine                         proline                         methionine                         leucine                         isoleucine    ______________________________________

The term "conditions of high stringency" refers to hybridization andwashing under conditions that permit binding (or hybridization) of anucleic acid molecule used for screening (where such nucleic acidmolecule typically encodes an inflammatory or therapeutic cardiomyopathypeptide or fragment thereof) such as an oligonucleotide probe or cDNAmolecule probe, to highly homologous DNA or RNA molecules. An exemplaryhigh stringency wash solution is 0.2×SSC and 0.1 percent SDS used at atemperature of between 50° C.-65° C.

Where oligonucleotide probes are used for hybridization, one of thefollowing two exemplary high stringency solutions may be used. The firstof these is 6×SSC with 0.05 percent sodium pyrophosphate at atemperature of 35° C.-62° C., depending on the length of theoligonucleotide probe. For example, 14 base pair probes can be washed at35-40° C., 17 base pair probes can be washed at 45-50° C., 20 base pairprobes can be washed at 52-57° C., and 23 base pair probes can be washedat 57-63° C. The temperature can be increased 2-3° C. where thebackground non-specific binding appears high. A second high stringencysolution utilizes tetramethylammonium chloride (TMAC) for washingoligonucleotide probes. One stringent washing solution is 3 M TMAC, 50mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature usingthis solution is a function of the length of the probe. For example, a17 base pair probe is washed at about 45-50° C.

As used herein, the term "effective amount" when used in conjunctionwith "inflammatory cardiomyopathy peptide", refers to the amount ofinflammatory cardiomyopathy peptide necessary to induce an immuneresponse in a patient, whether human or non-human, to which it isadministered.

The term "effective amount" when used in conjunction with "therapeuticcardiomyopathy peptide" refers to the amount of such peptide necessaryto decrease or prevent inflammatory cardiomyopathy in a patient, whetherhuman or non-human, to which it is administered.

The term "CpG oligodeoxynucleotide" refers to an oligodeoxynucleotidecontaining the internal motif "GACGTT". Preferably, the CpGoligodeoxynucleotide will be about 20 nucleotides in length, but mayrange from about 14 to 30 or more nucleotides in length.

The inflammatory cardiomyopathy peptides, fragments, and/or derivativesthereof, as well as the therapeutic cardiomyopathy peptides, fragments,variants, and derivatives thereof, may be prepared by chemical synthesismethods (such as solid phase peptide synthesis) using techniques knownin the art such as those set forth by Merrifield et al., (J. Am. Chem.Soc., 85: 2149 1963!), Houghten et al. (Proc Natl Acad. Sci. USA, 82:5132 1985!), and Stewart and Young (Solid Phase Peptide Synthesis,Pierce Chemical Co., Rockford, Ill. 1984!). Such peptides may besynthesized with or without a methionine on the amino terminus. Thesechemically synthesized inflammatory cardiomyopathy peptides andtherapeutic cardiomyopathy peptides or fragments, variants, andderivatives, may be acylated at the amino terminus using standardprocedures (see "Protection by Acylation" in: Bodanszky, Principles ofPeptide Synthesis, Springer-Verlag, N.Y. 1984! p. 85 et seq.). Apreferred acylation group is the acetyl group. Such peptides may beoxidized using standard methods to form disulfide bridges if desired.The inflammatory cardiomyopathy peptides and therapeutic cardiomyopathypeptides produced by chemical synthesis and optionally acylated areexpected to have biological activity comparable to inflammatorycardiomyopathy peptides or therapeutic cardiomyopathy peptides producedrecombinantly or purified from natural sources, and thus may be usedinterchangably with the corresponding recombinant or natural peptides.

Inflammatory cardiomyopathy peptides, therapeutic cardiomyopathypeptides, and fragments, variants, or derivatives thereof, can also beprepared using well known recombinant DNA technology methods such asthose set forth in Sambrook et al. (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1989!) and/or Ausubel et al., eds, (Current Protocols in MolecularBiology, Green Publishers Inc. and Wiley and Sons, NY 1994!).

A gene or cDNA encoding full length alpha myosin chain polypeptide, fulllength beta myosin chain polypeptide or fragments thereof which containthe DNA encoding the corresponding M7A-alpha or M7A-beta peptides of thepresent invention may be obtained for example by screening suitablegenomic or cDNA library (such as a human muscle tissue cDNA or genomiclibrary), or by PCR amplification. Probes or primers useful forscreening the library can be generated based on the sequences set forthherein, or based on information for other known genes or gene fragmentsfrom the same or a related family of genes, such as, for example,conserved motifs found in other related or homologous genes. Inaddition, where an alpha myosin chain protein, a beta myosin chainprotein, or fragment thereof (which contains the nucleic acid sequenceencoding an inflammatory cardiomyopathy peptide or therapeuticcardiomyopathy peptide) has been identified from one species, all or aportion of that gene may be used as a probe to identify homologous genesfrom other species, or to identify allelic variants of the same species.The probes or primers may be used to screen cDNA libraries from varioustissue sources believed to express the protein from which theinflammatory cardiomyopathy peptide or therapeutic cardiomyopathypeptide is derived (such as alpha or beta myosin chain genes, Chlamydiaouter membrane protein genes, etc). Typically, conditions of highstringency will be employed for screening to minimize the number offalse positives obtained from the screen. Once the full length gene orthe portion of the full length gene encoding an inflammatorycardiomyopathy peptide or the therapeutic cardiomyopathy peptide of thepresent invention has been obtained, restriction endonuclease digestionmay be used to obtain the portion of the gene encoding only the desiredcardiomyopathy peptide or fragment thereof. This gene fragment can thenbe inserted into a suitable expression vector (as described herein) toproduce the cardiomyopathy peptide.

Another means to prepare a gene encoding inflammatory cardiomyopathypeptide or fragment thereof, or therapeutic cardiomyopathy peptide orfragment thereof, or to prepare a CpG oligodeoxynucleotide of thepresent invention, is to employ chemical synthesis using methods wellknown to the skilled artisan such as those described by Engels et al.(Angew. Chem. Intl. Ed., 28: 716-734 1989!). These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of any of the inflammatory or therapeutic cardiomyopathypeptides. Nucleic acid variants may be produced using site directedmutagenesis, PCR amplification, or other appropriate methods, where theprimer(s) have the desired point mutations (see Sambrook et al., supra,and Ausubel et al., supra, for descriptions of mutagenesis techniques).Chemical synthesis using methods described by Engels et al., supra, mayalso be used to prepare such nucleic acid variants. Other methods knownto the skilled artisan may be used as well. Preferred nucleic acidvariants are those containing nucleotide substitutions accounting forcodon preference in the host cell that is to be used to produce thepeptide. Other preferred variants are those encoding conservative aminoacid changes as described above (e.g., wherein the charge or polarity ofthe naturally occurring amino acid side chain is not alteredsubstantially by substitution with a different amino acid) as comparedto wild type.

The gene, cDNA, or nucleic acid fragment encoding an inflammatorycardiomyopathy peptide or therapeutic cardiomyopathy peptide can beinserted into an appropriate expression or amplification vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thealpha or beta myosin chain peptide gene and/or expression of the genecan occur). The gene, cDNA or fragment thereof encoding an inflammatorycardiomyopathy peptide or therapeutic cardiomyopathy peptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether the peptide or fragment thereof is to be glycosylatedand/or phosphorylated. If so, yeast, insect, or mammalian host cells arepreferable.

Typically, the vectors used in any of the host cells will contain 5'flanking sequence (also referred to as a "promoter") and otherregulatory elements as well such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe peptide or polypeptide to be expressed, and a selectable markerelement. Each of these elements is discussed below. Optionally, thevector may contain a "tag" DNA sequence, i.e., an oligonucleotidemolecule located at the 5' or 3' end of the peptide coding sequence; theoligonucleotide molecule encodes polyHis (such as hexaHis), or other"tag" such as FLAG, HA (hemaglutinin Influenza virus) or myc for whichcommercially available antibodies exist. This tag is typically fused tothe peptide upon its expression, and can serve as an tag for affinitypurification of the peptide from the host cell. Affinity purificationcan be accomplished, for example, by column chromatography usingantibodies against the tag as an affinity matrix. Optionally, the tagcan subsequently be removed from the purified inflammatorycardiomyopathy peptide by various means such as using suitablepeptidases.

The 5' flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5' flanking sequences from more than one source), synthetic, or itmay be the native alpha or beta myosin chain polypeptide 5' flankingsequence. As such, the source of the 5' flanking sequence may be anyunicellular prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that the 5' flankingsequence is functional in, and can be activated by, the host cellmachinery.

The 5' flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically, 5'flanking sequences useful herein other than the native or endogenousflanking sequence will have been previously identified by mapping and/orby restriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full length nucleic acid molecule of the 5' flankingsequence may be known. Here, the 5' flanking sequence may be synthesizedusing the methods described above for nucleic acid synthesis or cloning.

Where all or only a portion of the 5' flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5' flanking sequence fragments from thesame or another species.

Where the endogenous 5' flanking sequence is not known, a fragment ofDNA containing a 5' flanking sequence may be isolated from a largerpiece of DNA that may contain, for example, a coding sequence or evenanother gene or genes. Isolation may be accomplished by restrictionendonuclease digestion using one or more carefully selected enzymes toisolate the proper DNA fragment. After digestion, the desired fragmentmay be isolated by agarose gel purification, Qiagen® column or othermethods known to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe alpha myosin chain (poly)peptide. If the vector of choice does notcontain an origin of replication site, one may be chemically synthesizedbased on a known sequence, and ligated into the vector.

The transcription termination element is typically located 3' of the endof the inflammatory cardiomyopathy peptide coding sequence and serves toterminate transcription of the inflammatory or therapeuticcardiomyopathy peptide. Usually, the transcription termination elementin prokaryotic cells is a G-C rich fragment followed by a poly Tsequence. While the element is easily cloned from a library or evenpurchased commercially as part of a vector, it can also be readilysynthesized using methods for nucleic acid synthesis such as thosedescribed above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3' to the promoter and 5' to the coding sequence of the alphamyosin chain peptide to be synthesized. The Shine-Dalgarno sequence isvaried but is typically a polypurine (i.e., having a high A-G content).Many Shine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for an inflammatory or therapeuticcardiomyopathy peptide to be secreted from the host cell, a signalsequence may be used to direct the peptide out of the host cell where itis synthesized. Typically, the signal sequence is positioned within thecoding region of the nucleic acid molecule encoding the inflammatory ortherapeutic cardiomyopathy peptide, or directly at the 5' end of theinflammatory or therapeutic cardiomyopathy peptide coding region. Manysignal sequences have been identified, and any of them that arefunctional in the selected host cell may be used in conjunction with thegene encoding the inflammatory or therapeutic cardiomyopathy peptide.Therefore, the signal sequence may be homologous or heterologous to thegene encoding the inflammatory or therapeutic cardiomyopathy peptide.Additionally, the signal sequence nucleic acid molecule may bechemically synthesized using methods set forth above.

In many cases, transcription of the gene encoding the inflammatory ortherapeutic cardiomyopathy peptide is increased by the presence of oneor more introns in the vector; this is particularly true where theinflammatory or therapeutic cardiomyopathy peptide is produced ineukaryotic host cells, especially mammalian host cells. The introns usedmay be naturally occurring within the gene from which the inflammatoryor therapeutic cardiomyopathy peptide is derived. Where the intron isnot naturally occurring, the intron(s) may be obtained from anothersource. The position of the intron with respect to the 5' flankingsequence and the gene encoding the inflammatory or therapeuticcardiomyopathy peptide is generally important, as the intron must betranscribed to be effective. As such, where the gene inserted into theexpression vector is a cDNA molecule, the preferred position for theintron is 3' to the transcription start site, and 5' to the polyAtranscription termination sequence. Preferably for cDNA, the intron willbe located on one side or the other (i.e., 5' or 3') of the cDNA suchthat it does not interrupt the this coding sequence. Any intron from anysource, including any viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this invention, provided thatit is compatible with the host cell(s) into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this invention are typicallyconstructed from starting vectors such as commercially availablevectors. Such vectors may or may not contain some of the elements to beincluded in the completed vector. If none of the desired elements arepresent in the starting vector, each element may be individually ligatedinto the vector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to "blunt" the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in "sticky ends" using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra.

Alternatively, two or more of the elements to be inserted into thevector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

One other method for constructing the vector to conduct all ligations ofthe various elements simultaneously in one reaction mixture. Here, manynonsense or nonfunctional vectors will be generated due to improperligation or insertion of the elements, however the functional vector maybe identified and selected by restriction endonuclease digestion of thefinal vectors.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, LaJolla,Calif.), pET15b (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding inflammatory or therapeutic cardiomyopathy peptide has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or peptideexpression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, cansynthesize inflammatory cardiomyopathy peptide which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). After collection, the peptide can be purified using methodssuch as molecular sieve chromatography, affinity chromatography, and thelike.

Selection of the host cell for inflammatory or therapeuticcardiomyopathy peptide production will depend in part on whether thepeptide is to be glycosylated or phosphorylated (in which caseeukaryotic host cells are preferred), and the manner in which the hostcell is able to "fold" the protein into its native tertiary structure(e.g., proper orientation of disulfide bridges, etc.) such thatbiologically active peptide is prepared by the cell. However, where thehost cell does not synthesize biologically active peptide, the peptidemay be "folded" after synthesis using appropriate chemical conditions asdiscussed below.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines, are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the peptides of the presentinvention.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14: 810-817 1993!), Lucklow (Curr. Opin.Biotechnol., 4: 564-572 1993!) and Lucklow et al. (J. Virol., 67:4566-4579 1993!). Preferred insect cells are Sf-9 and Hi5 (Invitrogen,Carlsbad, Calif.)

Insertion (also referred to as "transformation" or "transfection") ofthe vector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The culture medium will usually contain all nutrients necessary for thegrowth and survival of the cells. Suitable media for culturing E. colicells are for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM,all of which may be supplemented with serum and/or growth factors asrequired by the particular cell line being cultured. A suitable mediumfor insect cultures is Grace's medium supplemented with yeastolate,lactalbumin hydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of inflammatory or therapeutic cardiomyopathy peptideproduced in the host cell can be evaluated using standard methods knownin the art. Such methods include, without limitation, Western blotanalysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gelelectrophoresis, HPLC separation, immunoprecipitation, and/or activityassays such as DNA binding gel shift assays.

If the inflammatory or therapeutic cardiomyopathy peptide has beendesigned to be secreted from the host cells, the majority of peptide maybe found in the cell culture medium. Peptides prepared in this way willtypically not possess an amino terminal methionine, as it is removedduring secretion from the cell. If however, the peptide is not secretedfrom the host cells, it will be present in the cytoplasm and/or thenucleus (for eukaryotic host cells) or in the periplasm (for gramnegative bacteria host cells) and may have an amino terminal methionine.

For peptide situated in the host cell cytoplasm and/or nucleus, the hostcells are typically first disrupted mechanically or with detergent torelease the intra-cellular contents into a buffered solution. Thepeptide can then be isolated from this solution.

Purification of inflammatory or therapeutic cardiomyopathy peptide fromsolution can be accomplished using a variety of techniques. If thepeptide has been synthesized such that it contains a tag such asHexahistidine (cyclin E2/hexaHis) or other small peptide such as FLAG(Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen, Carlsbad,Calif.) at either its carboxyl or amino terminus, it may essentially bepurified in a one-step process by passing the solution through anaffinity column where the column matrix has a high affinity for the tagor for the polypeptide directly (i.e., a monoclonal antibodyspecifically recognizing alpha or beta myosin chain peptide). Forexample, polyhistidine binds with great affinity and specificity tonickel, thus an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of peptide/polyHis (see forexample, Ausubel et al., eds., Current Protocols in Molecular Biology,Section 10.11.8, John Wiley & Sons, New York 1993!).

Where the inflammatory or therapeutic cardiomyopathy peptide is preparedwithout a tag attached, and no antibodies are available, other wellknown procedures for purification can be used. Such procedures include,without limitation, ion exchange chromatography, molecular sievechromatography, HPLC, native gel electrophoresis in combination with gelelution, and preparative isoelectric focusing ("Isoprime"machine/technique, Hoefer Scientific). In some cases, two or more ofthese techniques may be combined to achieve increased purity.

If it is anticipated that the inflammatory or therapeutic cardiomyopathypeptide will be found primarily intracellularly, the intracellularmaterial (including inclusion bodies for gram-negative bacteria) can beextracted from the host cell using any standard technique known to theskilled artisan. For example, the host cells can be lysed to release thecontents of the periplasm/cytoplasm by French press, homogenization,and/or sonication followed by centrifugation.

If the inflammatory or therapeutic cardiomyopathy peptide has formedinclusion bodies in the periplasm, the inclusion bodies can often bindto the inner and/or outer cellular membranes and thus will be foundprimarily in the pellet material after centrifugation. The pelletmaterial can then be treated with a chaotropic agent such as guanidineor urea to release, break apart, and solubilize the inclusion bodies.The peptide in its now soluble form can then be analyzed using gelelectrophoresis, immunoprecipitation or the like. If it is desired toisolate the peptide, isolation may be accomplished using standardmethods such as those set forth below and in Marston et al. (Meth. Enz.,182: 264-275 1990!).

If inflammatory or therapeutic cardiomyopathy peptide inclusion bodiesare not formed to a significant degree in the periplasm of the hostcell, the peptide will be found primarily in the supernatant aftercentrifugation of the cell homogenate, and the peptide can be isolatedfrom the supernatant using methods such as those set forth below.

In those situations where it is preferable to partially or completelyisolate the inflammatory or therapeutic cardiomyopathy peptide,purification can be accomplished using standard methods well known tothe skilled artisan. Such methods include, without limitation,separation by electrophoresis followed by electroelution, various typesof chromatography (immunoaffinity, molecular sieve, and/or ionexchange), and/or high pressure liquid chromatography. In some cases, itmay be preferable to use more than one of these methods for completepurification.

Chemically modified inflammatory or therapeutic cardiomyopathy peptidecompositions (`derivatives`) are included within the scope of thepresent invention. The chemical moiety selected is typically watersoluble so that the alpha myosin chain peptide does not precipitate inan aqueous environment, such as a physiological environment. Thechemical moiety selected is usually modified to have a single reactivegroup, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled asprovided for in the present methods. The chemical moiety may be of anymolecular weight, and may be branched or unbranched. Included within thescope of inflammatory cardiomyopathy peptide moieties is a mixture ofsuch moieties. Preferably, for therapeutic use of the end-productpreparation, the moiety will be pharmaceutically acceptable.

The water soluble moiety or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxypolyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers or oligomers, poly-(N-vinyl pyrrolidone)polyethylene glycol, propylene glycol homopolymers, a polypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g.,glycerol) and polyvinyl alcohol.

For the acylation reactions, the polymer or oligomer(s) selected shouldhave a single reactive ester group. For reductive alkylation, thepolymer or oligomer(s) selected should have a single reactive aldehydegroup. A preferred reactive aldehyde is polyethylene glycolpropionaldehyde, which is water stable, or mono (C1-C10) alkoxy oraryloxy derivatives thereof (see U.S. Pat. No. 5,252,714).

Pegylation of inflammatory or therapeutic cardiomyopathy peptide may becarried out by any of the pegylation reactions known in the art, asdescribed for example in the following references: Focus on GrowthFactors 3: 4-10 (1992); EP 0 154 316; and EP 0 401 384. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer) as described below.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated inflammatorycardiomyopathy peptide will generally comprise the steps of (a) reactinga peptide with polyethylene glycol (such as a reactive ester or aldehydederivative of PEG) under conditions whereby the peptide becomes attachedto one or more PEG groups, and (b) obtaining the reaction product(s). Ingeneral, the optimal reaction conditions for the acylation reactionswill be determined based on known parameters and the desired result. Forexample, the larger the ratio of PEG: protein, the greater thepercentage of poly-pegylated product.

Generally, the peptide-polymer moieties of the present invention areuseful for the same purposes as those described herein for inflammatorycardiomyopathy peptide and therapeutic cardiomyopathy peptide molecules.However, the polymer/peptide molecules disclosed herein may haveadditional activities, such as enhanced or reduced biological activity,or other characteristics, such as increased or decreased half-life, ascompared to the non-derivatized molecules.

An alternative to chemical modification to produce peptide derivativesis production of fusion peptides. Such fusion peptides can be preparedby attaching polyamino acids to the peptides. For example, the polyaminoacid may be a carrier protein which serves to increase the half life orstability of the peptide such as serum albumin, in particular, humanserum albumin, an antibody or portion thereof (such as the antibodyconstant region known as "Fc", and, in particular, human or humanizedFc), or other polyamino acid. Preparation of such fusion peptides isreadily accomplished using known cloning and recombinant DNA methodssuch as those set forth herein.

The inflammatory cardiomyopathy peptides, therapeutic cardiomyopathypeptides, and fragments thereof, variants, and derivatives thereof, maybe employed alone, together, or in combination with other pharmaceuticalcompositions. Such peptides, fragments, variants, and derivatives may beused in combination with each other, and/or with cytokines, growthfactors, antibiotics, anti-inflammatories, and/or chemotherapeuticagents as is appropriate.

The therapeutic cardiomyopathy peptides, whether administered alone orin combination, may be useful as vaccines for preventing or decreasingboth autoimmune inflammatory cardiomyopathy and inflammatorycardiomyopathy due to Chlamydia or other bacterial or viral infectionsthat cause inflammatory cardiomyopathy.

Nucleic acid molecules encoding inflammatory or therapeuticcardiomyopathy peptides, fragments, and/or derivatives that do notthemselves encode peptides that are biologically active may be useful ashybridization probes in diagnostic assays to test, either qualitativelyor quantitatively, for the presence of Chlamydia spp. DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

The inflammatory or therapeutic cardiomyopathy peptides, fragments,variants, and/or derivatives may be used to prepare antibodies usingstandard methods. Thus, antibodies that react with the inflammatory ortherapeutic cardiomyopathy peptides, as well as reactive fragments ofsuch antibodies, are also contemplated as within the scope of thepresent invention. The antibodies may be polyclonal, monoclonal,recombinant, chimeric, single-chain and/or bispecific. Typically, theantibody or fragment thereof will either be of human origin, or will be"humanized", i.e., prepared so as to prevent or minimize an immunereaction to the antibody when administered to a patient. The antibodyfragment may be any fragment that is reactive with the inflammatory andtherapeutic cardiomyopathy peptides of the present invention, such as,F_(ab), F_(ab'), etc. Also provided by this invention are the hybridomasgenerated by presenting an inflammatory or therapeutic cardiomyopathypeptide or a fragment thereof as an antigen to a selected mammal,followed by fusing cells (e.g., spleen cells) of the mammal with certaincancer cells to create immortalized cell lines by known techniques. Themethods employed to generate such cell lines and antibodies directedagainst all or portions of a human alpha or beta myosin chain peptide ofthe present invention are also encompassed by this invention.

The antibodies may be used therapeutically. The antibodies may furtherbe used for in vivo and in vitro diagnostic purposes, such as in labeledform to detect the presence of Chlamydia in a body fluid or cell sample.

Preferred antibodies are human antibodies, either polyclonal ormonoclonal.

Various assays can be used to identify biologically active inflammatoryor therapeutic cardiomyopathy peptides. Such assays include, forexample, injection of one or more of such peptides into a mammal such asa mouse, followed by analysis of heart tissue for inflammatorycardiomyopathy. Such procedures are described in the Examples herein.

Pharmaceutical Compositions and Administration

Pharmaceutical compositions of the inflammatory and therapeuticcardiomyopathy peptides are within the scope of the present invention.Such compositions may comprise an effective amount of peptide,fragments, variants, or derivatives in admixture with a pharmaceuticallyacceptable carrier. The carrier material may be water for injection,preferably supplemented with other materials common in solutions foradministration to mammals. Typically, an inflammatory or therapeuticcardiomyopathy peptide compound will be administered in the form of acomposition comprising purified peptide, fragment, variant, orderivative in conjunction with one or more physiologically acceptablecarriers, excipients, or diluents. Neutral buffered saline or salinemixed with serum albumin are exemplary appropriate carriers. Preferably,the product is formulated as a lyophilizate using appropriate excipients(e.g., sucrose). Other standard carriers, diluents, and excipients maybe included as desired. Other exemplary compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefor.

The inflammatory or therapeutic cardiomyopathy peptide compositions canbe administered parenterally. Alternatively, the compositions may beadministered intravenously or subcutaneously. When systemicallyadministered, the compositions for use in this invention may be in theform of a pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such pharmaceutically acceptable peptide solutions, withdue regard to pH, isotonicity, stability and the like, is within theskill of the art.

Formulations of inflammatory or therapeutic cardiomyopathy peptidecompositions useful for practicing the present invention may be preparedfor storage by mixing the selected composition having the desired degreeof purity with optional physiologically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company 1990!) in the form of alyophilized cake or an aqueous solution. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients and are preferably inert atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight peptides or polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

An effective amount of the inflammatory or therapeutic cardiomyopathypeptide composition(s) to be employed will depend, for example, upon theobjectives such as the indication for which the peptide is beingadministered, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal effect. A typical daily dosage may range from about 0.1μg/kg to up to 100 mg/kg or more, depending on the factors mentionedabove. Typically, a clinician will administer the peptide compositionuntil a dosage is reached that achieves the desired effect. The peptidecomposition may therefore be administered as a single dose, or as two ormore doses (which may or may not contain the same amount of peptide)over time, or as a continuous infusion via implantation device orcatheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of indication under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The peptide composition to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes. Where the peptide composition is lyophilized,sterilization using these methods may be conducted either prior to, orfollowing, lyophilization and reconstitution. The composition forparenteral administration ordinarily will be stored in lyophilized formor in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of administration of the composition is in accord with knownmethods, e.g. oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice which may optionally involve the use of a catheter. Wheredesired, the compositions may be administered continuously by infusion,bolus injection or by implantation device.

Screening Assays

Included in the scope of the present invention are methods of screeninga mammal for susceptiblity to, or risk of, autoimmune orbacterial-induced inflammatory cardiomyopathy, comprising assaying abiological fluid obtained from the mammal for activated T-cells to,and/or antibodies against, any of the inflammaotry cardiomyopathypeptides of the present invention.

By way of example, an individual can be evaluated for circulatingantibodies, or T-cells that have been primed against either endogenousmyosin alpha chain peptides of the present invention (such antibodies orprimed T-cells, if present, could indicate an increased chance ofdeveloping autoimmune inflammatory cardiomyopathy), or to peptides fromChlamydia outer membrane proteins (as described in the Examples below;the presence of such circulating antibodies or primed T-cells couldindicate an increased chance of developing bacterial-inducedinflammatory cardiomyopathy). Such evaluation can be accomplished byobtaining a sample of blood from the individual, lysing the red bloodcells in the sample (using, for example, ammonium chloride), collectingwhite blood cells, and incubating the white blood cells with one or moreinflammatory cardiomyopathy peptides of the present invention in thepresence of radioactive thymidine or an equivalent thereof. The cellscan be harvested and counted for radioactivity. Higher levels ofradioactivity as compared with controls (where the controls compriseT-cells not primed against the peptides, i.e., not previously exposed tothe peptides) would correlate to an increased risk of developinginflammaotry cardiomyopathy. Those individuals at risk could then bede-sensitized through the use of vaccine therapy using one or moreinflammatory cardiomyopathy peptides of the present invention as anactive component of the vaccine.

In a second type of screening assay, the serum component of a bloodsample from an individual could be tested for the presence of antibodiesagainst the inflammatory cardiomyopathy peptide(s) of the presentinvention using ELISA, Western blot, or other suitable technique todetect antibodies. The presence of such antibodies could indicate andincreased risk of developing inflammatory cardiomyopathy, in which casethe individual could be made tolerant of the peptides via vaccinetherapy as described above.

Oligonucleotides as Adjuvants

Certain bacterial DNA molecules purportedly can have immunostimulatoryeffects in vivo and in vitro (Davis et al., J. Immunol. , 160: 870 -8761998!). However, prior to the present invention, it was not known thatcertain oligodeoxynucleotides having a CpG motif (GACGTT) could beuseful as adjuvants for vaccines.

As described in Example 2 below, it has been found that certainoligonucleotides, when injected simultaneously with an inflammatorycardiomyopathy peptide of the present invention, can serve to increasethe immunogenicity of the peptide. The sequences of sucholigodeoxynucleotides are set forth below:

GATTGCCTGACGTCAGAGAG (SEQ ID NO:10)

TCCATGACGTTCCTGATGCT (SEQ ID NO:11)

TCCATGACGTTCCTGACGTT (SEQ ID NO:12)

GTACTGACGTTTACTCTTGG (SEQ ID NO:13)

These oligodeoxynucleoitdes can be prepared using standard methods foroligonucleotide synthesis such as the phosphoramidite method asdescribed above (see also Marshall, W. S., Drug Discovery Today, 3:34-42 1998!). In some cases, phosphorothioate modification can increasethe in vivo stability. Such modification is well known in the art, andcan be accomplished using, for example, the procedures as described inStein et al., Nuc,. Acids Res., 16: 3209-3221 1988! or Caruthers,Science, 230: 281-285 1985!.

EXAMPLES EXAMPLE 1

Identification and Preparation of Peptides Inducing InflammatoryCardiomyopathy

A peptide from a region of the murine alpha myosin heavy chainpolypeptide (Genbank accession number M76598) was prepared usingstandard FMOC (fluorenylmethoxycarbonyl)/t-butyl based solid phasepeptide chemistry (Pummerer et al., J. Clin Invest., 97: 2057-20621996!). This peptide, referred to as "M7A-alpha", has the followingsequence:

SLKLMATLFSTYASAD (SEQ ID NO:2)

The DNA encoding this peptide is:

TCCCTCAAGCTCATGGCTACACTCTTCTCTACCTATGCTTCTGCTGAT (SEQ ID NO:23)

A fragment of this peptide, which contains amino acids 1-14, wasevaluated for its ability to induce autoimmune inflammatorycardiomyopathy as follows. Twenty seven Balb/c mice of about 6 weeks ofage were immunized twice on days 0 and 7 with Freund's complete adjuvant(FCA) containing about 100 microliters of about 500 micrograms/ml of thepeptide (prepared synthetically using standard peptide synthesisprocedures). The mice were analyzed for the presence of inflammatorycardiomyopathy 21 days after the initial injection. The mice weresacrificed, and the hearts were dissected out, treated with formalin,and paraffin embedded. The heart tissue was then sectioned into sectionsof approximately 4 micrometers thick. The sections were stained usingconventional procedures with hematoxylin (to detect nuclei) and eosin.Tissue sections were then viewed microscopically, and the mononuclearcells (which are completely absent in normal heart tissue) in eachsection were counted. In addition, each section was viewed forcardiomyocyte damage (necrosis). Severity of inflammatory cardiomyopathywas then determined taking into account both the tissue damage and theamount of mononuclear cell infiltration. The following scale was used toassign severity. A score of 1.0 indicates that up to five percent of thetissue was damaged/infiltrated; a score of 2.0 indicates that 5-10percent of the tissue was damaged/infiltrated; a score of 3.0 indicatesthat 10-20 percent of the tissue was damaged/infiltrated; and a score of4.0 indicates that more than 20 percent of the tissue wasdamaged/infiltrated.

Results of the analysis indicated that 24 of the 27 mice had developedinflammatory cardiomyopathy.

To test the specificity of the peptide of SEQ ID NO:2 to causeinflammatory cardiomyopathy, a second peptide derived from a homologousregion of the murine beta myosin heavy chain (referred to as"M7A-beta")was tested in an identical manner. This peptide has thefollowing amino acid sequence:

SLKLLSNLFANYAG (SEQ ID NO:3)

The DNA sequence encoding this peptide is:

TCCCTCAAGCTCCTAAGTAATCTGTTTGCCAACTATGCTGGA (SEQ ID NO:25)

Nineteen Balb/c mice of about 6 weeks of age were injected with thispeptide in a manner identical to that described above. Twenty one daysafter the initial injection, these mice were analyzed for inflammatorycardiomyopathy using the method described above, and none were found tohave developed the disease.

The sequences of SEQ ID NOS: 2 and 3 were compared to identify thoseamino acid residues necessary to induce inflammatory cardiomyopathy. Thefollowing "consensus sequence" was generated:

MAxxxS (SEQ ID NO:1)

The GCG (Wisconsin Package) program LOOKUP was used to generate a listfile of bacterial and viral sequences from the PIR public proteindatabase. The list file was then searched using SEQ ID NO:1 to identifythose bacterial and viral proteins containing this sequence motif.

Surprisingly, several peptides were found that are fragments of cysteinerich outer membrane proteins from various species of Chlamydia. Thesequences of these peptides and the corresponding DNA sequences are setforth below.

C. trachomatis cysteine-rich outer membrane protein 1:

VLETSMAEFTSTNVIS (SEQ ID NO:4)

C. trachomatis cysteine-rich outer membrane protein 2: VLETSMAESLSTNVIS(SEQ ID NO:5)

C. trachomatis cysteine-rich outer membrane protein 3: VLETSMAEFISTNVIS(SEQ ID NO:6)

C. pneumoniae cysteine-rich outer membrane protein: GIEAAVAESLITKIVA(SEQ ID NO:7)

C. psittaci cysteine-rich outer membrane protein: KIEAAAAESLATRFIA (SEQID NO:8)

C. trachomatis protein 11: MGSMAFHKSRLFLT (SEQ ID NO:9)

The respective DNA sequences of these peptides are as follows:

GTGTTAGAGACCTCTATGGCAGAGTTCACCTCTACAAACGTTATTAGC (SEQ ID NO:17)

GTGTTAGAGACCTCTATGGCAGAGTCTCTCTCTACAAACGTTATTAGC (SEQ ID NO:18)

GTGTTAGAGACCTCTATGGCAGAGTTTATCTCTACAAACGTTATTAGC (SEQ ID NO:19)

GGTATAGAGGCCGCTGTAGCAGAGTCTCTGATTACTAAGATCGTCGTC (SEQ ID NO:20)

AAGATAGAGGCCGCTGCTGCAGAGTCTCTTGCTACAAGATTCATTGCC (SEQ ID NO:21)

ATGGGCTCGATGGCTTTCCATAAAAGTAGGTTGTTCTTAACT (SEQ ID NO:22)

Each of the above peptides was prepared using the standard FMOCchemistry procedure (see above)and acetylated at the amino terminususing standard chemistry methods, and injected into mice (along with theM7A-alpha peptide as a control) to determine whether these peptidescould induce inflammatory cardiomyopathy. The procedure for injectionwas identical to that described above. The mice were sacrificed 21 daysafter the initial injection, and the hearts were dissected out andsectioned as described above.

The following scale was used to assign severity of inflammatorycardiomyopathy in response to peptide injection. A score of 1.0indicates that up to about five percent of the tissue wasdamaged/infiltrated; a score of 2.0 indicates that about 5-10 percent ofthe tissue was damaged/infiltrated; a score of 3.0 indicates that about10-20 percent of the tissue was damaged/infiltrated; and a score of 4.0indicates that more than about 20 percent of the tissue wasdamaged/infiltrated. The results are shown in Table 1. For mouseM7A-alpha, the complete sequence of SEQ ID NO:2 was used.

Table 1

                  TABLE 1    ______________________________________    Peptide      Prevalence  Percent Severity    ______________________________________    mouse M7A-alpha                 18/21       86%     2.9  0.7!    C. trach. 1 CRP                 12/15       80%     1.4  9.4!    C. trach. 2 CRP                 7/8         88%     1.3  0.5!    C. trach. 3 CRP                 7/8         88%     1.1  0.6!    C. pneum. CRP                  6/10       60%     1.1  0.2!    C. psitt. CRP                  5/10       50%     1.0  0!    C. trach. p11                 4/8         50%     1.0  0!    ______________________________________

The numbers in parentheses under "Severity" are standard deviations.

As can be seen, mouse M7A-alpha and the three C. trachomatis peptidesinduced the highest level of disease, and mouse M7A-alpha generated thegreatest severity of disease.

Separately, the human homolog of the murine M7A-alpha peptide wasevaluated using the procedure described above. The sequence of thispeptide is:

SLKLMATLFSSYAT (SEQ ID NO:16)

The DNA encoding this peptide has the following sequence:

TCCCTCAAGCTCATGGCCACTCTCTTCTCCTCCTACGCAACT (SEQ ID NO:24)

This human peptide was found to induce inflammatory cardiomyopathy toabout the same degree as murine M7A-alpha.

In a separate study, about 50-100 micrograms of both murine M7A-alphaand M7A-beta (SEQ ID Nos:2 and 3, respectively) peptides weresimultaneously injected into mice. The mice did not develop inflammatorycardiomyopathy, suggesting that the M7A-beta peptide protects the mammalfrom developing inflammatory cardiomyopathy. Such results indicate thatM7A-beta and related peptides (e.g., homologous peptides from otherspecies, such as human see SEQ ID NO:15! as well as conservativesubstitutions of these peptides) could be useful as vaccines to decreaseor prevent inflammatory cardiomyopathy. In addition, the peptides of SEQID NOS: 7, 8, 9, and/or conservative variants of SEQ ID NO: 3, as wellas the human homolog of murine M7A-beta, the sequence of which is setforth immediately below as SEQ ID NO:15, which have a lower incidence ofinducing inflammatory myocarditis or do not induce inflammatorymyocarditis at all can be used as vaccines.

LKLLSTLFANYAGA (SEQ ID NO:15)

The DNA encoding this peptide has the following sequence:

CTCAAGTTGCTCAGCACCCTGTTTGCCAACTATGCTGGGGCT (SEQ ID NO:26)

EXAMPLE 2

Oligodeoxvnucleotides as Immunostimulators

An oligonucleotides derived from the DNA encoding a 60 kDa cysteine richouter membrane protein from Chlamydia trachomatis (de la Maza et al.,Infect. Immun., 59: 1196-1201 1991!) containing a CpG motif and referredto as a "CpG oligo" (SEQ ID NO:13), and its counterpart not containingthe CpG motif, the "non-CpG oligo" (SEQ ID NO:14), were synthesizedusing standard phosphoramidite chemistry, and were phosphorothioatemodified (Stein et al., supra; Caruthers et al., supra). The sequence ofthese is set forth below:

GTACTGACGTTTACTCTTGG (SEQ ID NO:13)

GTACTGAGCTTTACTCTTGG (SEQ ID NO:14)

Balb/c mice were immunized at day 0 and day 7 with a solution containingabout 50 micrograms of M7A-alpha peptide (SEQ ID NO:2) and about 10 nmolof the oligodeoxynucleotide of either SEQ ID NO:13 (CpGoligodeoxynucleotide) or SEQ ID NO:14 (non-CpG oligodeoxynucleotide),together with Freund's incomplete adjuvant. Negative control micereceived about 10 nmol of the oligodeoxynucleotide of SEQ ID NO:13without peptide, and positive control mice received about 50 microgramsof M7A-alpha peptide with complete Freund's adjuvant.

After 21 days, the mice were analyzed for the presence and severity ofinflammatory heart disease as described in Example 1. The results areshown in Table 2 below.

                  TABLE 2    ______________________________________    Adjuvant Peptide       Prevalence                                    Severity    ______________________________________    CFA      M7A-alpha     5/5      3.8 ± 0.4    CPG      M7A-alpha     5/5      1.2 ± 0.4    non-CpG  M7A-alpha     1/5      1.0 ± 0.0    CpG      None          0/5      --    ______________________________________

Surprisingly, the CpG oligonucleotide plus M7A-alpha peptide inducedinflammatory heart disease in the absence of Freund's complete adjuvant,indicating that this oligonucleotide, which contains the CpG motif, canserve as a potent immunostimulator. The oligonucleotide containing thenon-CpG motif was hardly effective as an adjuvant. Other CpGoligodeoxynucleotides tested and found to be immunostimulatory includethe oligos set forth in SEQ ID Nos:10-12 (see above).

EXAMPLE 3

Assessing the Risk of Inflammatory Cardiomyopathy

About 10 ml of blood is obtained from a patient suspected of being atrisk for inflammatory cardiomyopathy. The blood is treated with ammoniumchloride to lyse red blood cells, after which the sample is centrifugedto pellet white blood cells. The white blood cells are collected andadded to about 2 ml of RPMI cell culture medium containing about 10percent fetal calf serum, and about 1-100 micrograms of M7A-alphapeptide or a Chlamydia peptide (as set forth in Example 1) are thenadded. The cell sample is then incubated at about 37 C. and about 5percent carbon dioxide for up to about 4 days in the presence oftritiated thymidine (about 1 micro Curie). The cells are then washed andcounted for radioactivity.

Samples from patients previously exposed to Chlamydia, or have aproclivity towards autoimmune inflammatory myocarditis will show ahigher level of T-cell proliferation as measured by higher levels ofradioactivity when compared to white blood cell samples from individualsnever exposed to Chalmydia or having no history of, nor proclivity to,autoimmune cardiomyopathy.

In an alternate procedure, blood from a patient can be obtained andcentrifuged to collect the plasma. The plasma can then be tested viastandard ELISA or other comparable procedure to assay for the presenceof antibodies in the plasma that recognize any of the inflammatorycardiomyopathy peptides such as M7A-alpha or the Chlamydia peptides.Standards for the ELIZA can include plasma from individuals neverexposed to Chalmydia or having no history of, nor proclivity to,autoimmune cardiomyopathy. When the sample serum antibody levels arecompared with the standard serum antibody levels, a higher level of theantibodies as compared with the control serum indicates an increasedrisk of inflammatory cardiomyopathy.

    __________________________________________________________________________    #             SEQUENCE LISTING    - <160> NUMBER OF SEQ ID NOS: 26    - <210> SEQ ID NO 1    <211> LENGTH: 6    <212> TYPE: PRT    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <221> NAME/KEY: UNSURE    <222> LOCATION: (3)..(5)    <223> OTHER INFORMATION: Xaa's at above positio - #ns can be any amino    acid.    <220> FEATURE:    <223> OTHER INFORMATION: Consensus sequence for my - #osin.    - <400> SEQUENCE: 1    - Met Ala Xaa Xaa Xaa Ser      1               5    - <210> SEQ ID NO 2    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Mouse    - <400> SEQUENCE: 2    - Ser Leu Lys Leu Met Ala Thr Leu Phe Ser Th - #r Tyr Ala Ser Ala Asp    #                 15    - <210> SEQ ID NO 3    <211> LENGTH: 14    <212> TYPE: PRT    <213> ORGANISM: Mouse    - <400> SEQUENCE: 3    - Ser Leu Lys Leu Leu Ser Asn Leu Phe Ala As - #n Tyr Ala Gly    #                 10    - <210> SEQ ID NO 4    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 4    - Val Leu Glu Thr Ser Met Ala Glu Phe Thr Se - #r Thr Asn Val Ile Ser    #                 15    - <210> SEQ ID NO 5    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 5    - Val Leu Glu Thr Ser Met Ala Glu Ser Leu Se - #r Thr Asn Val Ile Ser    #                 15    - <210> SEQ ID NO 6    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 6    - Val Leu Glu Thr Ser Met Ala Glu Phe Ile Se - #r Thr Asn Val Ile Ser    #                 15    - <210> SEQ ID NO 7    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Chlamydia pneumoniae    - <400> SEQUENCE: 7    - Gly Ile Glu Ala Ala Val Ala Glu Ser Leu Il - #e Thr Lys Ile Val Ala    #                 15    - <210> SEQ ID NO 8    <211> LENGTH: 16    <212> TYPE: PRT    <213> ORGANISM: Chlamydia psittaci    - <400> SEQUENCE: 8    - Lys Ile Glu Ala Ala Ala Ala Glu Ser Leu Al - #a Thr Arg Phe Ile Ala    #                 15    - <210> SEQ ID NO 9    <211> LENGTH: 14    <212> TYPE: PRT    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 9    - Met Gly Ser Met Ala Phe His Lys Ser Arg Le - #u Phe Leu Thr    #                 10    - <210> SEQ ID NO 10    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    <220> FEATURE:    #from the DNANFORMATION: An oligonucleotide derived    #outer membrane protein fromeine rich          Chlamydia trachomatis.    - <400> SEQUENCE: 10    # 20               agag    - <210> SEQ ID NO 11    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    <220> FEATURE:    #from the DNANFORMATION: An oligonucleotide derived    #outer membrane protein fromeine rich          Chlamydia trachomatis.    - <400> SEQUENCE: 11    # 20               tgct    - <210> SEQ ID NO 12    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    <220> FEATURE:    #from the DNANFORMATION: An oligonucleotide derived    #outer membrane protein fromeine rich          Chlamydia trachomatis.    - <400> SEQUENCE: 12    # 20               cgtt    - <210> SEQ ID NO 13    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    <220> FEATURE:    <223> OTHER INFORMATION: Oligonucleotide derived from - # the DNA    #outer membrane protein fromeine rich          Chlamydia trachomatis containing a C - #pG motif and referred to as          CpG oligo.    - <400> SEQUENCE: 13    # 20               ttgg    - <210> SEQ ID NO 14    <211> LENGTH: 20    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    <220> FEATURE:    #from the DNANFORMATION: An oligonucleotide derived    #outer membrane protein fromeine rich          Chlamydia trachomatis which does not - # contain the CpG motif and          referred to as a non-CpG oligo.    - <400> SEQUENCE: 14    # 20               ttgg    - <210> SEQ ID NO 15    <211> LENGTH: 14    <212> TYPE: PRT    <213> ORGANISM: Human    - <400> SEQUENCE: 15    - Leu Lys Leu Leu Ser Thr Leu Phe Ala Asn Ty - #r Ala Gly Ala    #                 10    - <210> SEQ ID NO 16    <211> LENGTH: 14    <212> TYPE: PRT    <213> ORGANISM: Human    - <400> SEQUENCE: 16    - Ser Leu Lys Leu Met Ala Thr Leu Phe Ser Se - #r Tyr Ala Thr    #                 10    - <210> SEQ ID NO 17    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 17    #                48tggc agagttcacc tctacaaacg ttattagc    - <210> SEQ ID NO 18    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 18    #                48tggc agagtctctc tctacaaacg ttattagc    - <210> SEQ ID NO 19    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 19    #                48tggc agagtttatc tctacaaacg ttattagc    - <210> SEQ ID NO 20    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Chlamydia pneumoniae    - <400> SEQUENCE: 20    #                48tagc agagtctctg attactaaga tcgtcgtc    - <210> SEQ ID NO 21    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Chlamydia psittaci    - <400> SEQUENCE: 21    #                48ctgc agagtctctt gctacaagat tcattgcc    - <210> SEQ ID NO 22    <211> LENGTH: 42    <212> TYPE: DNA    <213> ORGANISM: Chlamydia trachomatis    - <400> SEQUENCE: 22    #  42              tcca taaaagtagg ttgttcttaa ct    - <210> SEQ ID NO 23    <211> LENGTH: 48    <212> TYPE: DNA    <213> ORGANISM: Mouse    - <400> SEQUENCE: 23    #                48ctac actcttctct acctatgctt ctgctgat    - <210> SEQ ID NO 24    <211> LENGTH: 42    <212> TYPE: DNA    <213> ORGANISM: Human    - <400> SEQUENCE: 24    #  42              ccac tctcttctcc tcctacgcaa ct    - <210> SEQ ID NO 25    <211> LENGTH: 42    <212> TYPE: DNA    <213> ORGANISM: Mouse    - <400> SEQUENCE: 25    #  42              gtaa tctgtttgcc aactatgctg ga    - <210> SEQ ID NO 26    <211> LENGTH: 42    <212> TYPE: DNA    <213> ORGANISM: Human    - <400> SEQUENCE: 26    #  42              ccct gtttgccaac tatgctgggg ct    __________________________________________________________________________

We claim:
 1. A peptide selected from the group consisting of: SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:16.
 2. The peptide of claim 1 wherein theamino-terminal amino acid is acylated.
 3. The peptide of claim 2 whereinan acetyl group is used for acylation.
 4. A peptide selected from thegroup consisting of: SEQ ID NO:3 and SEQ ID NO:15.
 5. The peptide ofclaim 4 wherein the amino-terminal amino acid is acylated.
 6. Thepeptide of claim 5 wherein an acetyl group is used for acylation.
 7. Avaccine to decrease inflammatory cardiomyopathy comprising a peptide, anadjuvant, and an excipient, wherein the peptide consists of any of SEQID NOS; 2, 3, 4, 5, 6, 7, 8, 9, 15, or 16.